Comparative insight into nucleotide excision repair components of Plasmodium falciparum

DNA Repair 28 (2015) 60–72

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DNA Repair journal homepage: www.elsevier.com/locate/dnarepair

Comparative insight into nucleotide excision repair components of Plasmodium falciparum Leila Tajedin, Masroor Anwar, Dinesh Gupta, Renu Tuteja ∗ Malaria Group, International Centre for Genetic Engineering and Biotechnology, P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India

a r t i c l e

i n f o

Article history: Received 23 April 2014 Received in revised form 27 January 2015 Accepted 10 February 2015 Available online 20 February 2015 Keywords: DNA repair Helicase Malaria parasite Plasmodium falciparum Unwinding

a b s t r a c t Nucleotide excision repair (NER) is one of the DNA repair pathways crucial for maintenance of genome integrity and deals with repair of DNA damages arising due to exogenous and endogenous factors. The multi-protein transcription initiation factor TFIIH plays a critical role in NER and transcription and is highly conserved throughout evolution. The malaria parasite Plasmodium falciparum has been a challenge for the researchers for a long time because of emergence of drug resistance. The availability of its genome sequence has opened new avenues for research. Antimalarial drugs like chloroquine and mefloquine have been reported to inhibit NER pathway mediated repair reactions and thus promote mutagenesis. Previous studies have validated existence and implied possible association of defective or altered DNA repair pathways with development of drug resistant phenotype in certain P. falciparum strains. We conjecture that a compromised NER pathway in combination with other DNA repair pathways might be conducive for the emergence and sustenance of drug resistance in P. falciparum. Therefore we decided to unravel the components of NER pathway in P. falciparum and using bioinformatics based approaches here we report a genome wide in silico analysis of NER components from P. falciparum and their comparison with the human host. Our results reveal that P. falciparum genome contains almost all the components of NER but we were unable to find clear homologue for p62 and XPC in its genome. The structure modeling of all the components further suggests that their structures are significantly conserved. Furthermore this study lays a foundation to perform similar comparative studies between drug resistant and drug sensitive strains of parasite in order to understand DNA repair-related mechanisms of drug resistance. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Genome integrity is continuously challenged by DNA lesions and an individual cell can undergo up to one million DNA changes per day [1]. Both prokaryotic and eukaryotic organisms have evolved a rigorous system of checks and balances through the DNA repair machinery to maintain this genome integrity. DNA repair processes including nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR) and non-homologous end-joining (NHEJ) exist in both prokaryotic and eukaryotic organisms, and many of the proteins involved are highly conserved throughout evolution [2]. NER is more complex in eukaryotes as compared to prokaryotes but the general principle is same. During NER, the proteins assemble to recognize, incise, and excise the damaged strand from the genomic DNA [2]. Generally NER removes bulky and cross linked DNA adducts that

∗ Corresponding author. Tel.: +91 11 26741358; fax: +91 11 26742316. E-mail address: [email protected] (R. Tuteja). http://dx.doi.org/10.1016/j.dnarep.2015.02.009 1568-7864/© 2015 Elsevier B.V. All rights reserved.

result in distortion of the double helix structure of DNA, caused by both exogenous factors (such as chemicals and UV) and endogenous factors (oxidizing reactive species). NER pathway can be divided into two related subpathways – global genome repair (GGNER), which removes lesions from all regions of the genome and transcription-coupled repair (TC-NER), which repairs the damage from the transcribed strands of active genes [3]. The multifunctional cellular transcription initiation factor IIH (TFIIH) is involved in NER as well as transcription [4–6]. The mammalian TFIIH includes a core, containing the seven subunits XPB, XPD, p62, p52, p44, p34, and p8/TTD-A coupled to the cdkactivating kinase module (CAK) composed of the three subunits Cdk7, cyclin H, and MAT1 [4,7–10]. The XPD (RAD3) helicase plays the role of bridging between the core and CAK complex [4]. Various studies have shown that mutations in NER components result in rare disorders. Mutations in Xeroderma pigmentosum group B (XPB), Xeroderma pigmentosum group D (XPD), ERCC1XPF, XPG and p8 (also known as TF2H5 and TTDA) subunits cause autosomal recessive disorders, such as trichothiodystrophy (TTD), Xeroderma pigmentosum (XP), Cockayne’s syndrome (CS), Fanconi

L. Tajedin et al. / DNA Repair 28 (2015) 60–72

anemia and phenotypically heterogeneous forms of inherited disorder [7,11,12]. Malaria still has huge impact on human health and it is the world’s second biggest killer after tuberculosis [13]. For more than a century, scientists have tried to eradicate or control malaria, but still about 627,000 human deaths worldwide annually with an estimated infection rate of 207 million cases per year have been reported [14]. Among five Plasmodium species that cause human malaria, Plasmodium falciparum is the most deadly species which has developed resistance to antimalarial drugs such as chloroquine, sulfadoxine-pyrimethamine and even artemisinin [14–16]. The emergence of resistance in P. falciparum depends on multiple factors for instance the mutation rate of the parasite and the fitness costs associated with the resistance mutations. An increased mutation rate is certainly advantageous for adapting to adverse environments caused by introduction of drug [16,17]. In human tumor cells, bacteria and some other organisms DNA repair pathways including NER, BER, MMR, HR and NHEJ have been linked to increased mutation rates and drug resistance [18–20]. Some reports which have shown that BER, MMR and DNA doublestrand break repair (DSBR) pathways are somehow linked to drug resistance in P. falciparum and some of the proteins involved in these pathways can be the new anti-malarial drug targets [21–24]. Several non-synonymous single nucleotide polymorphisms (SNPs) in the MMR genes of artemisnin drug resistant strains in P. falciparum have been reported [15]. Previous studies have shown that antimalarial drugs including chloroquine, mefloquine, quinine and halofantrine inhibit the repair of UV light-induced DNA damage. So it has been proposed that altered DNA repair, either through defective repair mechanisms or drug-mediated inhibition, may be considered as the accelerator of drug resistance in the parasite [25]. TFIIH has role in transcription via interaction with RNA polymerase II. It has been reported that the inhibition of enzymatic activities of human XPB lead to the inhibition of RNA polymerase II-mediated transcription and likely NER [26]. Therefore the clinical symptoms observed in patients are most likely due to DNA repair defects and transcription deficiencies [7]. There are reports which suggest that transcriptional regulations are important in the control of gene expression in various life cycle stages of P. falciparum [27,28]. Any disturbance in the activity of components of

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TFIIH complex will most likely affect the development and growth of the parasite. The present study was undertaken in order to find the homologues of NER components in P. falciparum. We have also done a comparative analysis of these components with the human host (Homo sapiens) and yeast (Saccharomyces cerevisiae). Overall here we have reported the bioinformatics based in silico analysis of all the components of NER from P. falciparum. Using these approaches we were unable to identify the homologues of two proteins (p62 and XPC) in P. falciparum. This report has set the foundation for further comparative analysis of NER components from drug resistant and susceptible parasite strains to understand the mechanism of drug resistance. In addition it will be worthwhile to biochemically characterize all the components in order to investigate their function in the malaria parasite.

2. Materials and methods For this study, gene information was obtained from NCBI (http:// www.ncbi.nlm.nih.gov) and Plasmodb (http://plasmodb.org/ plasmo/). We have used different softwares and online servers for bioinformatics analysis, which are listed (Table S1). We used three species for bioinformatics based comparison and the PlasmoDB numbers and PDB numbers of templates are provided (Table 1). The downloaded sequences were used as query to match with the human homologue using BLAST search (www.ncbi.nlm.nih.gov). The corresponding human sequence was retrieved and various domains were manually assigned. Similarly the domains were also assigned manually in P. falciparum sequence and the data are presented in figures. The domain analysis was done using Scan Prosite at (http://expasy.org). Likewise the structural modeling was done using the swissmodel or phyre2 server. The molecular graphic images were produced using the UCSF Chimera package/PyMOL molecular graphic system. The comparative analysis of genome of three species was done through multiple sequence alignment using http://www.ebi.ac.uk/Tools/msa/clustalw2/. The protein–protein interaction analysis of different subunits of NER components of P. falciparum was done through http://string-db.org/ and the human homologues of the interacting

Table 1 PlasmoDB numbers and PDB accession numbers. Protein

Plasmodium gene ID

Molecular weight (kDa)

PDB ID of template (chain)

Percentage identity ∼

Modeled residue range

Figure no.

XPB XPD p62 p52 p44

PF3D7 PF3D7 None PF3D7 PF3D7

1037600 0934100 1244200 1314900

102.87 122.83 None 111.87 45.98

p34 p8 Cyclin H CDK7 MAT1 Centrin-2 ERCC1/Rad10 XPF XPG

PF3D7 PF3D7 PF3D7 PF3D7 PF3D7 PF3D7 PF3D7 PF3D7 PF3D7

1353500 1441900 1463700 1014400 0512300 0107000 0203300 1368800 0206000

38.32 7.8 39.23 37.98 31.30 19.6 28.2 204.53 178.7

49 27 None 19.178 36 19 23 32 18 42 28 83 34 36 23 and 25

15 16 17 18 19 20 21 22

FBL3/Rad7 LIG1 RAD23 RPA1 RPA2 RPA3 XPA XPC

PF3D7 PF3D7 PF3D7 PF3D7 PF3D7 PF3D7 PF3D7 None

1123200 1304100 1011700 0409600 0904800 1442100 0710400

93.3 104.5 44.3 70 36 44 43.8 None

4kt1 (A) 2hiv (A) 1oqy (A) 1jmc 1jmc 4gnx 1xpa None

18 28 22 35 30 25 26 None

576–806 20–1021 None 841–907 341–397 90–260 20–221 1–58 13–294 7–309 1–65 21–168 46–174 48–165 5–130 and 1207–1424 476–773 175–896 1–387 683–919 10–189 1–109 211–319 None

1 2 None 3 4 & S1

6 7 8 9 10 11 12 13 14

4ernA 2VSF (A) None 3domC 1z60 (A) 2X5N (A) 4PN7 (A) 2jnj (B) 1JKW 1V0 (B) 1G25 (A) 3kf9 (A) 2a1i (A) 2jpd 1rxw (A)

S. no. 1 2 3 4 5

S2 S3 S4 S5 S6 W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 None

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partners identified through PlasmoDB were further verified using http://asia.ensembl.org/index.html. For convenience, we divided figures of this work into three parts, TFIIH figures are presented in text and supplementary figures and figures for other components involved in NER are provided in one web link, http://203.92.44.100/Nuc-exp/webpagenew.htm and web link has been referred as ‘W’ in text. 3. Results and discussion In the present study we have reported the comparative analysis of almost all the components of NER from P. falciparum. Out of 22 proteins which are known to be involved in NER machinery in

human/yeast, ten components build up the TFIIH complex. Overall 20 NER proteins were detectable from P. falciparum using described approaches while nine out of twenty are part of TFIIH complex. Three components XPB, XPD and p52 from P. falciparum show low similarity to human and yeast counterparts due to several insertions. The comparative analysis of the helicase components XPB, XPD and their interacting partners p52 and p44 is presented as main Figs. 1–4. The comparative analysis of rest of the TFIIH complex components such as p34, p8 and the CAK complex (cyclin, CDK7 and MAT1) is presented as supplementary Figs. 1–6. Based on these results a hypothetical model for all the TFIIH components of P. falciparum is presented as Fig. 5. We have also performed the comparative analysis of all the other NER components such

Fig. 1. (A) Comparison of amino acid sequence of P. falciparum (Pf) XPB with H. sapiens (Hs) and S. cerevisiae (Sc). The alignment was done using clustalw2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The plasmodb number for P. falciparum XPB is PF10 0369 and the accession numbers for Hs and Sc sequences are P19447.1 and EEU05444.1 respectively. The conserved motifs are boxed in blue and the name of each motif (from I to VI) is written in roman numerals. NLS region in PfXPB is marked in violet. The green and red boxes show the ATP binding domain and helicase C terminal domain, respectively. (B) Structural modeling of XPB (i) Template, (ii) PfXPB model and (iii) Superimposed.

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Fig. 2. (A) Comparison of amino acid sequence of P. falciparum (Pf) XPD with H. sapiens (Hs) and S. cerevisiae (Sc). The plasmodb number for P. falciparum XPD sequence is PF3D7 0934100 and the accession numbers for Hs and Sc sequences are NP 000391 and NP 011098, respectively. The conserved motifs are boxed in blue color and the name of each motif (from I to VI) is written in roman numerals. NLS region in PfXPD is marked in purple color. Helicase C terminal domain is marked in red box. Green boxes are showing conserved cysteines. (B) Structural modeling of XPD (i) Template, (ii) PfXPD model and (iii) Superimposed.

as centrin, ERCC1, ERCC4/XPF, ERCC5/XPG, FBL3, ligase 1, Rad23, RPA1, RPA2, RPA3 and XPA and the data are presented as web Figs. 1–11. The detailed descriptions for all the above analysis are presented in the following sections. 3.1. Xeroderma pigmentosum B (XPB) XPB is a 3 –5 ATP dependent helicase that has been studied in human for its role in diseases like XP, TTDA and CS. XPB is a ∼89 kDa and ∼95 kDa protein in human and yeast respectively. In addition to its seven helicase motifs, one of them being the ATP binding site, XPB possesses two additional ‘ATPase’ motifs: the well-conserved R-E-D residue loop motif and the positively charged thumb (ThM) region. These motifs are specifically involved in the

regulation of the DNA-dependant ATPase activity of XPB and help to stabilize the binding of TFIIH to the damaged DNA, to allow the recruitment of other NER factors [29]. The P. falciparum homologue of XPB (103 kDa) (Table 1) is relatively larger in size as compared to its human counterpart. PfXPB contains nuclear localization signal (NLS) at its N-terminus (Fig. 1A, violet box). The comparison of PfXPB to HsXPB shows that it contains 104 additional amino acid residues, the helicase ATP binding domain and helicase C-terminal domain (Fig. 1A). The scanprosite analysis shows that there are several sites in PfXPB viz. N-glycosylation, cAMP and cGMP-dependent protein kinase phosphorylation, protein kinase C phosphorylation, casein kinase II phosphorylation, tyrosine kinase phosphorylation, Nmyristoylation and amidation. The 3D structural modeling of the

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Fig. 3. (A) Comparison of amino acid sequence of P. falciparum (Pf) p52 with H. sapiens (Hs) and S. cerevisiae (Sc). The plasmodb number for P. falciparum p52 sequence is PF3D7 1244200 and the accession numbers for Hs and Sc sequences are NP 001508.1 and Q02939.1, respectively. The NLS is boxed in violet, the red and green boxes denote asparagines and aspartic acid rich regions respectively. (B) Asparagine and aspartic acid rich regions in Pfp52. (C) Structural modeling of Pfp52 (i) Template, (ii) Pfp52 model and (iii) Superimposed.

PfXPB was done based on the known crystal structure of human XPB C-terminal (Table 1) [30]. The modeled structure of PfXPB and the template superimposedonly partially (Fig. 1B(i–iii)). The protein interaction analysis of PfXPB in the database shows variety of interacting partners for PfXPB. It is quite notable that PfXPB interacts with different polymerases too (Table 2). 3.2. Xeroderma pigmentosum D (XPD/RAD3/ERCC2) XPD also known as basic transcription factor 2, 80 kDa subunit (BTF2 p80), and xeroderma pigmentosum group D-complementing protein, ERCC-2 is the other subunit of TFIIH. Due to the presence of ssDNA-dependent ATPase and 5 –3 direction DNA helicase activities in XPD, it has been postulated that it plays an important role in NER [31]. XPD can regulate Cdk-activating kinase subcomplex and

can maintain the stability of the complex for transcription initiation [32]. PfXPD (Table 1) contains all the conserved helicase signature motifs as reported earlier also (Fig. 2A) [33]. The alignment results show that there is a typical difference in protein size of XPD in P. falciparum as compared to two other species and the molecular weight of protein is about 36 kDa more (Fig. 2A). The NLS sequence in P. falciparum is in the middle from amino acid 493–532, but this NLS and in fact this part of protein is absent in the human and yeast homologues (Fig. 2A, purple box). Most conserved sequences and motifs are located in the N and C terminal of XPD in all three species. There are several insertions in PfXPD. In addition, N terminal of PfXPD contains DEAD-2 family, a conserved region within a number of RAD3-like DNA-binding. The helicase C-terminal domain is located from amino acid 819–995 in PfXPD which is the

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Fig. 4. Structural modeling of Pfp44. (A) This structure covered VWA domain in N-terminal of Pfp44 (i) Template, (ii) Pfp44 (iii) Superimposed. (B) This structure covered zinc finger domain in C-terminal of Pfp44 (i) Template, (ii) Pfp44 (iii) Superimposed.

second of two tandem AAA domains found in a wide variety of helicases (Fig. 2A, red box). Interestingly, there is an insertion that contains a repetitive sequence of asparagine after NLS in PfXPD, which is absent in other species (Fig. 2A). The role of asparagine has been proposed to be in capping and it also provides key sites for N-linked glycosylation and modification of the protein chain. The study on Sulfolobus acidocaldarius XPD showed presence of four cysteine ligands and an iron–sulfur (Fe–S) cluster domain in XPD which is required for its helicase activity [34]. The cysteine ligands are conserved in all three species (Fig. 2A, green boxes), but we were unable to predict the Fe–S domains in PfXPD, HsXPD and ScXPD using bioinformatics approaches and therefore the Fe–S domains in PfXPD, HsXPD and ScXPD were assigned manually. The structural modeling of PfXPD was done using the known crystal structure of XPD from Thermoplasma acidophilum as the template (PDB ID 2VSF chain A) [35]. When the modeled structure of PfXPD and the template were superimposed, all seven signature motifs

and C-terminal helicase domain is completely superimposed and rest of the structure only partially superimposed (Fig. 2B(i–iii)). The interacting partner analysis for PfXPD shows that it interacts with a number of proteins such as polymerases, p44, p34 and Rad25 (human XPB homologue) (Table 2). The interaction of PfXPD and Pfp44 in database is in agreement with our experimental work [36].

3.3. p62/GTF2H1 The human p62 subunit or GTF2H1 is a 62 kDa polypeptide and it is homologous to Tfb1 in S. cerevisiae. In human, p62 plays an important role in architecture and function of TFIIH [37]. The p62/Tfb1 subunit is essential for both the transcription and DNA repair activities of TFIIH [37]. Using the methods described it was not possible to obtain the corresponding clear homologue for p62 for P. falciparum from the Plasmodb database.

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finger ring domains are known to participate in nucleic acid binding and protein–protein interactions. Several important sites such as: N-glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation and Nmyristoylation sites were also found. The structural modeling of Pfp44 was done using the known crystal structure of the RPN10 VWA domain in Schizosaccharomyces pombe (PDB ID 2X5N) [42] and solution structure of the C-terminal domain of Hsp44 (PDB ID 1z60) as the templates (Table 1) [43]. The first model and its template superimposed well in VWA domain site of N-terminus of Pfp44 (Fig. 4A(i–iii)). While the second modeled structure of Pfp44 and the template from Hsp44 partially superimposed in zinc finger domain site in C-terminus of Pfp44 (Fig. 4B(i–iii)), which suggests that the zinc finger domain might be different among these two species. As expected, the interacting partner analysis for Pfp44 protein showed high interaction score with XPD (Table 2). 3.6. p34/BTF2 p34

Fig. 5. The figure depicts the proposed hypothetical model for P. falciparum TFIIH showing its subunit interaction in the complex.

3.4. p52/GTF2H4/Tfb2 p52 (52 kDa) also known as GTF2H4 is another prominent subunit of TFIIH and it is homologous to Tfb2 in S. cerevisiae. During NER TTDA/p8 stimulates the ATPase activity of XPB through a direct interaction with p52 [38]. The study of Pfp52 (Table 1) shows that it has significantly evolved and contains a large insertion of asparagines and aspartic acid in its sequence (Fig. 3A, red and green boxes respectively, Fig. 3B). Such a long insertion has made its size almost double as compared to Hsp52 and Scp52 (Fig. 3A). The NLS sequence of Pfp52 is located nearly in the middle of the protein (Fig. 3A, violet box). The Pfp52 contains various sites for tyrosine kinase phosphorylation, N-myristoylation and amidation. The structural modeling of Pfp52 was done using the known crystal structure of p52 in S. cerevisiae as template (PDB ID 3dom, chain C) [38]. The modeled structure of Pfp52 and the template superimposed only with C terminal of Pfp52 (Fig. 3C(i–iii)). The result of interacting partner analysis for Pfp52 shows that it interacts with a number of hypothetical proteins (Table 2). 3.5. p44 (SSL1) Basic transcription factor 2, 44 kDa subunit is a homologue of SSL1 (suppressor of stem-loop protein 1) in S. cerevisiae [39]. p44 is the component of the core-TFIIH and is also involved in transcription by RNAP II [40]. N-terminus of p44 interacts with and regulates the activity of XPD whereas an intact C-terminus is required for a successful escape of RNAP II from the promoter [40]. Hsp44 is 395 amino acid protein [39] and Pfp44 is 401 amino acid protein (Table 1). p44 contains two domains VWA (Von Willebrand factor type A) and TFIIH C1-like domain in all species (Fig. S1B–D). The carboxyl-terminal region of p44 is essential for transcription activity. This region binds three zinc atoms through two independent domains. Two different types of zinc finger motifs are present, zinc finger C2H2 type domain in Hsp44 and Ssl1 (Fig. S1D) and zinc finger ring type domain in Pfp44 (Fig. S1B). A zinc finger is a small protein structure motif that is characterized by the coordination of one or more zinc irons in order to stabilize the fold [41]. The zinc

GTFIIH3 gene belongs to the TFB4 superfamily and encodes the protein, which is also known as basic transcription factor 2, 34 kDa subunit (BTF2 p34) [41]. p34 also possesses zinc finger domains which might aid binding of BTF2 to nucleic acids. BTF2 p34 is one of the 6 subunits forming the core-TFIIH, which associates with the CAK complex [40]. Both p44 and p34 possess zinc finger domains that may mediate BTF2 binding to nucleic acids [39]. The size of Pfp34 (Table 1) is almost similar to Hsp34 and TFB4. The number of conserved residues between p34 of all the species is relatively low (Fig. S2A). Tfb4 family is involved in the initiation of transcription and NER and is present in all three species (Fig. S2B–D). The sequence homology in p44 binding region of Pfp34 is relatively low compared to human and yeast. The C-terminal region of Pfp34 is rich in cysteine and zinc finger binding motif shows the high level of conservation (Fig. S2A). The scan prosite analysis revealed several important sites such as: N-glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation and tyrosine kinase phosphorylation sites. The structural modeling of Pfp34 was done with available structure of the p34 N-terminal domain from Chaetomium thermophilum (PDB ID 4PN7) (Table 1) [44]. VWA domain like fold has been reported in Ctp34 [44]. Although we were unable to find the vWA domain due to low sequence similarity but the structure model superimposition with template suggests that Pfp34 contains vWA domain which in both species superimposed significantly (Fig. S2E(i–iii)). PfXPB and PfXPD helicases are present in protein interaction prediction analysis of Pfp34 (Table 2). 3.7. p8/GTF2H5/TTDA/TFB5 The smallest subunit p8 of TFIIH, also known as GTF2H5 (general transcription factor IIH subunit 5), TTDA, TFB5 ortholog or TGF2H5, is a 71 amino acid nuclear protein that belongs to the TFB5 family. p8 has been shown to be required for DNA opening during NER by stimulating the ATPase activity of XPB through direct interaction with p52 [10]. Pfp8 (Table 1) is almost similar in size to its human and yeast counterparts (Fig. S3A). This protein contains the protein kinase C phosphorylation site. The structural modeling of Pfp8 was done using the known solution structure of the Hsp8 as the template (PDB ID 2jnj, chain B) [45]. The modeled structure of Pfp8 and the template superimposed partially (Fig. S3B(i–iii)). The protein interaction prediction analysis of Pfp8 showed interactions with TATA binding protein and the polymerase (Table 2). 3.8. Kinase subunits The CAK (cyclin-dependent kinase (CDK)-activating kinase) complex is composed of cyclin H, CDK7 and MAT1. CDKs are well

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Table 2 Predicted protein–protein interactions of TFIIH complex of P. falciparum. S. no. 1

Name and plasmodb no.

Plasmodb no. of interacting partners

Name of interacting partners

Human homologue as reported on PlasmoDB

XPB PF3D7 1037600

PFI1650w

DNA excision-repair helicase, putative Hypothetical protein Cysteine-tRNA ligase, putative

ENSP00000375809 (XPD) – ENSP00000369897 (cysteine tRNA ligase) ENSP00000314949 (DNA-directed RNA polymerase II)

PF11 0493-1 PF10 0149 PFC0805w

PF07 0115 ACC1

PF10 0023 PF13 0341 PfSUMO

2

TFIIH basal transcription factor subunit

PF10 0369

Helicase, putative

Cyc-1 MAL13P1.76-1

Rad51

Cyclin, putative. TFIIH basal transcription factor subunit cdk7, putative CDK-activating kinase assembly factor, putative DNA repair endonuclease, putative Hypothetical protein, conserved Hypothetical protein, conserved Hypothetical protein, conserved Rad51 homolog, putative

p62

None

None

p52 PF3D7 1244200

MAL13P1.76-1

TFIIH basal transcription factor subunit Hypothetical protein, conserved DNA excision-repair helicase, putative DNA repair protein RAD50, putative; Essential component of the MRN complex. DNA repair exonuclease, putative. CDK-activating kinase assembly factor, putative. CDK7, putative. Transcription initiation factor TFiid, TATA-binding protein; Gbph2

ENSP00000274400 (p44) ENSP00000228955 (p34) ENSP00000375809 (XPD) ENSP00000265335

ENSP00000375809 (XPD) ENSP00000228955 (p34) –

PF10 0369

DNA excision-repair helicase, putative Hypothetical protein, conserved Hypothetical protein, conserved CDK-activating kinase assembly factor, putative CDK7, putative DNA repair endonuclease, putative Helicase, putative

cyc-1

Cyclin, putative

XPD PF3D7 0934100

PFB0265c PF13 0279 PFL2125c MAL13P1.23

4

PF13 0279 PFI1650w PFF0285c

PFA0390w PFE0610c PF10 0141 PFE0305w gbph2 5

ENSP00000349877 (Cation transporting ATPase) ENSP00000341044 (Biotin carboxylase subunit of acetyl CoA carboxylase) – ENSP00000215587 (DNA-directed RNA polymerase 2) ENSP00000380990 (Ubiquitin-like protein) ENSP00000274400 (TFIIH basal transcription factor p44)

MAL13P1.76-

PF10 0141 PFE0610c

3

DNA-directed RNA polymerase II, putative; DNA-dependent RNA polymerase catalyzes the transcript Cation transporting ATPase, cation transporter Biotin carboxylase subunit of acetyl CoA carboxylase, putative Hypothetical protein DNA-directed RNA polymerase 2, putative Ubiquitin-like protein, putative

p44 PF3D7 1314900

PFI1650w PF13 0279 PFL2125c PFE0610c PF10 0141 PFB0265c

ENSP00000285398 (XPB) – ENSP00000274400 (p44) – ENSP00000261245 (CDK-activating kinase, MAT1) ENSP00000347978 (XPG) ENSP00000228955 (p34) – – ENSP00000216024 (Rad51)

ENSP00000385614 (MRE11) ENSP00000261245 (CDK-activating kinase, MAT1) – ENSP00000247219 (TATA-binding protein) –

ENSP00000261245 (CDK-activating kinase, MAT1) – ENSP00000347978 (XPG) ENSP00000285398 (XPB) –

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Table 2 (Continued) S. no.

6

Name and plasmodb no.

p34 PF3D7 1353500

Plasmodb no. of interacting partners

Name of interacting partners

Human homologue as reported on PlasmoDB

PF14 0081

DNA repair helicase, putative

MAL13P1.134-1

Helicase, putative

ENSP00000322287 (Rad3) ENSP00000402325 –

PFE0610c

CDK-activating kinase assembly factor, putative DNA excision-repair helicase, putative TFIIH basal transcription factor subunit Helicase, putative

PFI1650w MAL13P1.76-1 PF10 0369 PFL2125c cyc-1 PF10 0141 PFA 0525w PFE0305w PFC0805w 7

p8 PF3D7 1441900

PFE0305w PFC0805w

8

Cyclin PF3D7 1463700

PF10 0141 PFI1650w PFE0610c PF13 0279 PF10 0369

10

– – – ENSP00000247219 (TATA-binding protein) ENSP00000314949 (DNA-directed RNA polymerase II)

Transcription initiation factor TFiid, TATA-binding protein DNA-directed RNA polymerase II

ENSP00000247219 (TATA-binding protein) ENSP00000314949 (DNA-directed RNA polymerase II)

cdk7, putative DNA excision-repair helicase, putative CDK-activating kinase assembly factor, putative Hypothetical protein, conserved Helicase, putative

– ENSP00000375809 (XPD) ENSP00000261245 (CDK-activating kinase, MAT1) ENSP00000228955 (p34) ENSP00000285398 (XPB) ENSP00000314949 (DNA-directed RNA polymerase II) – –

PfPK6 CRK2

DNA-directed RNA polymerase II, putative; Pf protein kinase 6 P. falciparum Protein Kinase 5; Plays role in the control of eukaryotic cell cycle. I

CDK7 PF3D7 1014400

Cyc-1

Cyclin, putative

–

MAT1 PF3D7 0512300

PF10 0141 cyc-1 PF13 0279

CDK7, putative Cyclin, putative Hypothetical protein, conserved DNA excision-repair helicase, putative Helicase, putative

– – ENSP00000228955 (p34) ENSP00000375809 (XPD) ENSP00000285398 (XPB) ENSP00000274400 (p44) –

PFC0805w

9

Hypothetical protein, conserved Cyclin, putative CDK7, putative Transcription initiation factor TFIIB, putative Transcription initiation factor TFiid, TATA-binding protein DNA-directed RNA polymerase II, putative

ENSP00000261245 (CDK-activating kinase, MAT1) ENSP00000375809 (XPD) ENSP00000274400 (p44) ENSP00000285398 (XPB) –

PFI1650w PF10 0369 MAL13P1.76-1 PFL2125c PFE0870w PF14 0416 PFB0265c

known to co-ordinate the progression of cell cycle, DNA replication and transcription. It has been shown that the core and CAK subcomplexes are associated by the XPD helicase subunit, which in turn interacts with MAT1 of the CAK subcomplex or p44 of the core, respectively [7]. It has been reported that the C terminus of MAT1 binds to the cdk7-cyclin H complex and activates the cdk7 kinase activity [46].

TFIIH basal transcription factor subunit Hypothetical protein, conserved Transcriptional regulator, putative Hypothetical protein DNA repair endonuclease, putative

ENSP00000216297 (CDC68) ENSP00000360497 (Ring finger protein) ENSP00000347978 (XPG)

3.8.1. Cyclin H In a previous study the authors were not able to find the homologue of cyclin H in P. falciparum [47]. Here using BLAST analysis the homologue for cyclin H in P. falciparum was obtained. PfCyclinH (Table 1) shows ∼17% homology to the human and yeast cyclin (Fig. S4A). The 3D structural modeling of the PfCyclinH was done using the known crystal structure of HscyclinH as the template (PDB ID

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1JKW) [48]. The modeled structure of PfCyclinH and the template superimposed partially (Fig. S4B(i–iii)). The interacting partners for PfCyclinH were identified and the results show that it interacts with only two proteins such as XPD, cdk7 and a protein kinase (Table 2). 3.8.2. CDK7 In a previous study the authors reported several Cdk7 putative homologs with lower identity values but they were unable to find an exact homologue for PfCDK7 [47]. Using BLAST analysis we report a homologue for CDK7 in P. falciparum. PfCDK7 (Table 1) shows ∼37–40% homology to the yeast and human cdk7 (Fig. S5A). The domain analysis of all the CDK7 revealed that all of these contain the characteristic protein kinase domain (Fig. S5B–D). The 3D structural modeling of the PfCDK7 was done using the known crystal structure of PfPK5as the template (PDB ID 1V0B) [49]. When the modeled structure of PfCDK7 and the template were superimposed, it is clear that these structures superimpose partially (Fig. S5E(i–iii)). The interacting partners for PfCDK7 were identified and the results show that it interacts with only one protein cyclin in the database (Table 2). 3.8.3. MAT1 PfMAT1 (Table 1) shows ∼21–23% homology to the human and yeast MAT1 homologues (Fig. S6A). The domain analysis of all the MAT1 showed that all of these contain the characteristic Zinc finger RING-type signature motifs, which is cysteine rich [50] (Fig. S6B–D) and only HsMAT1 showed the presence of ubiquitin-interacting motif [51] of about 20 amino acid residues which recognizes ubiquitin (Fig. S6C). The 3D model of PfMAT1 was generated using solution structure of the N-terminal domain of the HsMAT1 (PDB ID 1G25) [52]. The PfMAT1 model and template superimposed in zinc finger domain site significantly (Fig. S6E(i–iii)). The results of the interacting partner prediction for PfMAT1 show that it interacts with at least ten proteins like XPD, p44 and Cdk7 (Table 2). The predicted protein–protein interactions analysis for all the components of TFIIH shows that the interacting partners for these components are significantly conserved between the parasite and human host (Table 2). The proposed hypothetical model as analyzed using different bioinformatics tools for P. falciparum TFIIH and its subunits arranged in a complex has been shown in Fig. 5. PfXPB, PfXPD, p52, p44 along with p34 form the core. The Pfp8 subunit might act as a regulatory protein interacting both with PfXPB or PfXPD and up regulating the helicase activity. MAT1, which shows good interaction with the core component, might get phosphorylated with PfCdk7 precomplexed to PfcyclinH of the CAK complex (Fig. 5). Other components involved in NER are described in the following sections. 3.9. Centrin-2 (CDC31) Centrin-2/CENT2 is a component of XPC complex and it is involved in GG-NER. CENT-2 along with RAD23B recruits and stabilizes XPC [53,54]. PfCENT2 (Table 1) is similar in size to its human and yeast counterparts (Fig. W1a) and contains four EF-hand calcium binding motifs which are highly conserved among eukaryotes (Fig. W1b B–D). This kind of domain consists of a 12 residues loop flanked on both sides by a 12 residue alpha-helical domain. EFhands undergo a conformational change upon binding calcium ions [55]. The structure model of PfCENT2 was generated using centrin from Scherffelia dubia (PDB ID 3kf9) as template. All four EF-hand calcium binding motifs in PfCENT2 model superimposed quite well with same motifs in the template (Fig. W1b E(i–iii)). Database analysis shows PfCENT2 can interact with RCC1 (guanidine nucleotide exchange factor) (Table W1).

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3.10. DNA excision repair protein ERCC-1 (Rad10) ERCC1 has a dramatic function in NER and mutations in ERCC1 result in cerebrooculofacioskeletal syndrome in humans [56]. ERCC1 has been shown to play important role in stabilizing and enhancing the functionality of the XPF endonuclease. ERCC-1 is the homologue of Rad10 in yeast. The size of PfERCC1 (Table 1) is similar to HsERCC1 and Rad10 (Fig. W2a A). The 3D structure model of PfERCC1 was generated using HsERCC1 as template (PDB ID 2A1I) [57]. When PfERCC1 model was superimposed with template the results show both structures covered binding domain and superimposed significantly (Fig. W2b E(i–iii)). The protein interaction prediction analysis showed that this protein can interact with at least seven proteins involved in DNA repair (Table W1). 3.11. Xeroderma pigmentosum complementation group F (XPF)/ERCC4 XPF is a DNA repair endonuclease encoded by the ERCC4 gene [58]. XPF functions in the removal of UV-C photoproducts and bulky adducts from DNA. The ERCC1-ERCC4 heterodimer is involved in DNA binding and additional protein–protein interactions [59]. PfXPF (Table 1) is almost double in size as compared to its HsXPF and contains repeated stretch of asparagines (Fig.W3a–c A). In all species, the conserved ERCC4 domain is located in C-terminal of the protein (Fig. W3d B). The structural modeling of PfXPF was done using the known crystal structure of HsERCC1 as the template (PDB ID 2JPD) [60] and PfXPF model superimposed with template partially (Fig. W3d C(i–iii)). The interacting partners for PfXPF were identified and the results show that it interacts with significant endonucleases of P. falciparum (Table W1). 3.12. Xeroderma pigmentosum complementation group G (XPG)/ERCC5 Human XPG encoded by the ERCC5 gene is a single stranded endonuclease involved in 3 incision during NER pathway [11]. RAD2 is human XPG homologue in S. cerevisiae. Sequence alignment of PfXPG (Table 1) with HsXPG and RAD2 revealed that the similarities are largely confined to two regions. The first being the XPGN domain, located at the N-terminal region from amino acid 1–126 in P. falciparum (Fig. W4a). (Fig. W4a). The second region is XPG internal (XPGI) domain and corresponds to the 1220–1289 amino acids that include highly conserved residues (Fig. W4b). The amino acids between N- and I-regions are not conserved. Helix-hairpin-helix class 2 (HhH2) is a motif from amino acid 1291 to 1324 (Fig. W4c B–D). This motif has two helices connected by a short turn and is present in DNA repair enzymes and DNA polymerases. The structure of PfXPG was modeled using structure of flap endonuclease-1 from Archaeoglobus fulgidus (PDB ID – 1RXW) as a template [61]. The template and model superimposed only partially in XPGN and XPGI domains (Fig. W4c E(i–iii)). We were unable to find any interacting protein using database approaches. 3.13. FBL3 (Leucine rich repeat protein) (RAD7) RAD7 is the homologues of HsFBL3 in S. cerevisiae and plays a crucial role in the repair of transcriptionally inactive DNA [62]. The size of PfFBL3 (Table 1) is almost two times as compared to HsFBL3. In human and yeast, this protein contains leucine rich repeat (LRR) motifs (Fig. W5b). The size of LRRs is mostly 20–30 amino acids and it can repeat several times from 2 to 42. The repeat number is 11 in HsFBL3 (LRR11) and 4 in RAD7 but we were unable to find any significant domain or motif in PfFBL3. There is an F-box domain in HsFBL3 which is not present in RAD7 and PfFBL3. The structural modeling of PfFBL3 was done using human protein structure (PDB

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ID 4KT1) as template [63]. Generated model and template superimposed partially (Fig. W5b D(i–iii)). Protein interaction analysis of PfFBL3 revealed that it can interact with phosphatase, guanylyl cyclase and some putative proteins (Table W1). 3.14. DNA ligase 1 (CDC9) DNA ligase1 contains two main domains one in N terminal and another in the C terminal of protein and the ATP dependent DNA ligase domain is located in the middle of the protein [64]. All three domains are present in PfLIG1, CDC9 and HsLIG1, but it is interesting to note that the PFLIG1 contains an insertion of about 100 amino acids between the N terminal and ATP dependent DNA ligase domain (Fig. W6a A; b, B–D). The structure model of PfLIG1 was generated using LIG1 (PDB ID 2HIV) as template [65]. The model and template superimposed partially and the insertion in PfLIG1 looped out (Fig. W6b E(i–iii)). The results of protein interaction analysis showed that PfLIG1 has ten partners including proliferating cell nuclear antigen (PCNA) (Table W1). 3.15. RAD23 Rad23 is an important protein with a crucial role in proteasomal degradation of misfolded proteins as well as DNA repair. Rad23 has two homologs in human which are known as Rad23A (encodes UV excision repair protein RAD23 homolog A) and Rad23B which is also known by the name: HR23B complements hHR23B [66]. Alternatively Rad23B is also named as Xeroderma pigmentosum group C (XP-C) repair-complementing complex of 58 kDa protein. It is a component of the protein complex that specifically corrects the NER defect of XP-C cell extracts in vitro. PfRAD23 is similar in size to its human and yeast counterparts (Fig. W7a A) and all of these contain one ubiquitin-like domain (UBQ) in N terminal and two ubiquitin associated (UBA) domains located in the middle and C terminal (Fig. W7b B–D). The 3D structure model of PfRAD23 was generated using human hHR23a (PDB ID 1OQY, chain A) as template. PfRad23 model and template superimposed in all three predicted domains partially (Fig. W7b E(i–iii)). Protein interaction analysis of PfRAD23 revealed that it can interact with ten proteins with helicase and protease activity (Table W1). 3.16. Replication protein A (RPA) RPA also known as replication factor A is a heterotrimeric protein, which is involved in DNA replication, homologous recombination, and NER, showing its crucial role in NER upon interaction with XPA [4–6,8]. Trimer subunits are: RPA1 (70 kDa), RPA2 (36 kDa) and RPA3 (14 kDa). The interaction of XPA with both RPA32 and RPA70 is indispensable for NER reactions [67]. The P. falciparum genome shows two genes as homologues of RPA. The larger gene is a homologue of RPA1 and the smaller gene is homologue of RPA 2 (Table 1). PfRPA1 shows a large insertion of ∼300 amino acids at the N-terminal region with repeats of arginine (Fig. W8a A). PfRPA1 contains all the conserved domains similar to its human and yeast counterparts except replication factor A domain (Fig. W8b B). The structure of PfRPA1 was modeled using the single-stranded-DNA-binding domain of human RPA as the template (PDB ID 1jmc, chain A) [68]. The modeled structure of PfRPA1 and the template superimposed in tRNA-anti codon domain partially (Fig. W8 C(i–iii)). The interacting partner analysis for PfRPA1 shows that it interacts with several proteins of repair system (Table W1). PfRPA2 is larger in size as compared to its human and yeast counterparts and shows ∼30% identity to the human RPA (Fig. W9a A). The domain search analysis revealed that primarily two domains are conserved in all three species (Fig. W9b B). The 3D structural

modeling of the PfRPA2 was done using the known crystal structure of its human homologue as the template (PDB ID 1jmc) [68]. The structure model only covered the tRNA-anti codon domain. The modeled structure of PfRPA2 and the template superimposed partially (Fig. W9b C(i–iii)). The interacting partners of PfRPA2 are listed in Table W1. PfRPA3 is similar in size (Fig. W10a A) and contains a replication factor A domain, which is conserved in all three species (Fig. W10b B). The 3D structural modeling of the PfRPA3 was done using the known crystal structure of RPA homologue from Ustilago maydis as the template (PDB ID 4gnx). The modeled structure of PfRPA3 and the template superimposed partially (Fig. W10b C(i–iii)). Interacting partner analysis of PfRPA3 revealed some interacting partners including thrombospondin-related anonymous protein (TRAP) (Table W1). 3.17. Xeroderma pigmentosum complementation group A (XPA) Human XPA is a zinc metalloprotein containing a C4-type zinc finger motif and forms a part of core incision complex of the NER system [4]. Recent studies on XPA have shown its function in verifying DNA damage, stabilizing repair intermediates and recruiting other NER factors to the damaged DNA site [69]. For NER, XPA is imported from cytoplasm to nucleus in response to cellular DNA damage response during S-phase [67]. PfXPA (Table 1) is larger in size as compared to its human counterpart but no domains were detectable in PfXPA (Fig. W11a A, B). The structural modeling of the PfXPA was done using the known crystal structure of HsXPA as the template (PDB ID 1xpa, chain A) [70]. The modeled structure of PfXPA and the template covered the domains in C terminus of protein and superimposed partially (Fig. W11b C(i–iii)). The interacting partners for PfXPA were identified in database and are listed in Table W1. 3.18. Xeroderma pigmentosum complementation group C (XPC) The yeast counterpart of human XPC is named as RAD4. The XPC protein has been shown to form a complex with HHR23B, which is one of the two human homologs of yeast NER protein, RAD 23 [5]. Functionally XPC is a key factor in GG-NER where it recognizes the damaged DNA and recruits the repair machinery [8]. Several domains have been found in XPC: a DNA binding domain, an hHR23B binding domain, centrin 2 binding domain and a TFIIH binding domain. Using BLAST analysis we were unable to find a clear homologue of human XPC in P. falciparum database PlasmoDB. Our results show that almost all the NER components are present in P. falciparum genome but we were unable to identify clear homologue of p62 and XPC in its genome. The analysis of protein–protein interactions in database further indicates that interacting partners for almost all these components are significantly conserved between the parasite and human host. 4. Conclusions Multiple factors such as properties of drug, its mode of action and host response are involved in development of drug resistance. It has been suggested that altered or defective DNA repair pathways including NER might be one such factor which may help an organism to acquire mutations that can lead to development of drug resistant phenotypes under drug pressure. This might be occurring in P. falciparum as suggested by previous studies [25,71]. Therefore taking advantage of these reports, which indirectly implicated presence of a defective NER pathway in drug resistant parasites, we used in silico methods to study the NER components in parasite. Overall in the present study we have reported that almost all the components of NER machinery are present in P. falciparum genome

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but we were unable to identify the clear homologues of p62 and XPC using the bioinformatics approaches. One of the probable reasons for our inability to find the clear homologues in P. falciparum for p62 and XPC could be due to their low sequence homology to the human/yeast counterparts. This analysis revealed that some of the NER components (XPD, XPF, XPG, p52, RPA1 and RPA2) in P. falciparum contain large insertions of asparagines or aspartic acid residues. The overall identity for some components between P. falciparum, human and yeast was low but the domains and signature motifs are significantly conserved. Interestingly we were unable to identify the domains in PfFBL3 but structural similarity was significant to the human counterpart. The predicted protein–protein interactions analysis for all the NER components shows that the interacting partners for these components are significantly conserved between the parasite and human host. Emergence of drug resistant parasites is a major challenge to control malaria and it has been well established that some antimalarial drugs inhibit the repair reactions in the parasite. There is still a huge gap in understanding various repair mechanisms in the malaria parasites. Further work on the biochemical and functional characterization of various components of NER complex will shed light on how P. falciparum is able to perform all the NER related functions. As stated previously some of the NER proteins contain large insertions therefore it will be interesting to work on these evolutionary divergent features and discover the role of these insertions in their function. The studies reported here lay a foundation that most likely NER machinery exists in P. falciparum and further comparative mutation and single nucleotide polymorphisms studies of NER components between the drug resistant and drug susceptible strains will aid in understanding and unraveling the causes of antimalarial drug resistance. These observations can further help to identify novel drug targets and ultimately design novel drugs to combat malaria. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The work in RT’s laboratory is partially supported by Department of Biotechnology and Department of Science and Technology grants. Infra-structural support from the Department of Biotechnology, Government of India is gratefully acknowledged. The authors sincerely thank the two reviewers for constructive and useful comments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. dnarep.2015.02.009. References [1] H. Lodish, A. Berk, S. Zipursky, P. Matsudaira, D. Baltimore, J. Darnell, Molecular Cell Biology, W.H. Freeman, New York, 2000. [2] O.D. Scharer, Chemistry and biology of DNA repair, Angew. Chem. Int. Ed. 42 (2003) 2946–2974. [3] T. Lindahl, R.D. Wood, Quality control by DNA repair, Science 286 (1999) 1897–1905. [4] L. Schaeffer, V. Moncollin, R. Roy, A. Staub, M. Mezzina, A. Sarasin, G. Weeda, J.H. Hoeijmakers, J.M. Egly, The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor, EMBO J. 13 (1994) 2388–2392. [5] S. Iben, H. Tschochner, M. Bier, D. Hoogstraten, P. Hozak, J.M. Egly, I. Grummt, TFIIH plays an essential role in RNA polymerase I transcription, Cell 109 (2002) 297–306. [6] S.N. Guzder, P. Sung, V. Bailly, L. Prakash, S. Prakash, RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription, Nature 369 (1994) 578–581.

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